CN101012453A - CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof - Google Patents
CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof Download PDFInfo
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Abstract
The invention discloses a CHO cellar strain of high-effective expressive exogenous rhBMP2, which is characterized by the following: adopting CHO-dhfr- in the dihydrofolic acid reductase flaw-typed Chinese hamster ovary cell as host cell; infecting exogenous hBMP2 plasmid; adopting secretory expressive product to induce myoblast system C2C12 to display the activity to stimulate sclerotomal cell; obtaining single-clone strain of CHO (rhBMP-2) with the content of BMP2 at 7.83ug/24h*106; fitting for clinic treatment.
Description
Technical field
The invention belongs to the genetic engineering pharmaceutical technical field, relate to a kind of recombinaant CHO cell CHO (rhBMP-2) and establishment method thereof that efficiently expresses human bone morphorgenetic protein 2.Specifically, be the immortalization mammal cell line that makes up stable transfection human bone morphorgenetic protein 2 (hBMP2) gene coded sequence, after pressure screening and gene amplification, obtain clonal cell line stable, highly effective expressing rhBMP 2.
Background technology
Owing to reasons such as wound, infection, tumour and heteroplasia make bone lose some sclerotin, symptom such as the intractable bone of formation is damaged, nonunion and fracture delayed union is one of difficult problem thorny in the clinical practice.Treatment bone damaged best method is an autologous bone transplanting, but except exist the source limited, get that the bone district has that complication takes place may, the reparation speed of autologous bone transplanting is comparatively slow, thereby greatly limits it and be extensive use of.Allogenic bone transplantation has been let alone naturally.The factor of seeking promote osteogenesis and suitable bone transplantation substitute material mix treat that different clinically reasons cause that bone is damaged, diseases such as nonunion and fracture delayed union, be that the target of effort is mutually finished in domestic and international many laboratories, seek and set up external a large amount of acquisitions human bone morphorgenetic protein 2 stable and biologically active is domestic and international very active research field always always.
Clinical bone transplantation substitute material commonly used does not have bone-inducting active mostly at present, only has the artificial or natural materials of bone conduction and support effect.If with these materials and osteogenic factor-Delicious peptide (BMP) with efficient bone-inducting active, combine according to the organizational project principle, make it both have bone-inducting active, have bone conduction and support effect again, can improve the value for clinical application that bone is transplanted repair materials greatly.
1889, Senn found that the ground substance of bone of decalcification can promote the reparation that bone is damaged.Nineteen thirty, Levander is expelled to the ethanol crude extract of bone in the mouse tissue, and discovery can be induced new bone forming.1961, Sharrard and Collins went calcium to be used for the treatment of children's spinal fusion from the body bone with disodium salt.Nineteen sixty-five, Urist has found in ground substance of bone and can induce osteoplastic active substance, and with this active substance called after " Delicious peptide ".Nineteen eighty-three, Reddi and Sampath confirm to have that to induce bone forming active be protein ingredient rather than ground substance of bone itself in the ground substance of bone.1988, Johnson and its colleague carried out the damaged clinical trial of people BMP treatment bone with purifying.
At present, found more than ten kind of Delicious peptide, wherein Delicious peptide 2,6,7 has stronger bone inductive effect.Particularly BMP-2 has the activity of very strong induced osteogenesis, can induce bone forming separately in vivo, therefore has been applied to union of fracture, and spinal fusion is in numerous clinical applications such as plastic surgery and dental tissue engineering.
Homology is very high between the different Delicious peptide molecules, all synthetic to comprise nitrogen end signal peptide, propetide and carbon teminal mature peptide precursor forms, after processing is modified, cut signal peptide and preceding peptide moiety after, mature peptide partly forms glycosylated homology or heterodimer, induces bone forming.But natural B MP tissue content is low, the source is limited, purification procedures is loaded down with trivial details, the cost costliness, and have untoward reactions such as infection, allergy, be difficult to satisfy the needs of clinical practice.
The production for treating that is established as of gene recombination technology has been opened up new way with albumen.Can pass through gene recombination technology now, prepare people BMP in a large number, be used for the damaged treatment that waits relative disease of clinical bone.Because the limitation of prokaryotic cell prokaryocyte heterologous gene expression system self is difficult to overcome, the eukaryotic cell heterologous gene expression system is widely used in expressing the biological activity protein that needs posttranslational modification, particularly mammalian expression system has post transcriptional modificaiton function accurately, compare with the yeast cell to express system with the procaryotic cell expression system, the glycosylated protein of expressing near the native protein molecule, is the first-selected system that present recombinant glycoprotein is produced aspect molecular structure, physicochemical property and biological function.
In different mammalian cell expression systems; Chinese hamster ovary cell (CHO) exogenous protein expression system uses through practice for many years; become the mammal cell line of the most successful expression foreign protein; its advantage is that foreign gene can be stably integrated in the cell chromosome; and be easy to mass-producing and cultivate, these two characteristics are that exogenous protein large-scale preparation has been created condition.Wherein Tetrahydrofolate dehydrogenase (DHFR) defective type Chinese hamster ovary celI lacks the endogenous Tetrahydrofolate dehydrogenase, in the time of will containing the expression vector cotransfection of dihydrofolate reductase gene and goal gene, Tetrahydrofolate dehydrogenase (DHFR) gene and goal gene are integrated in the same site of host chromosome usually, competitive inhibitor at DHFR: under the condition that aminomethyl petrin (MTX) exists, thereby the increase of DHFR gene copy number drives the purpose that reaches raising destination gene expression amount.
Show at Chinese patent pertinent data result for retrieval that at present by the Chinese hamster ovary celI with people BMP2 and DHFR gene co-transfection DHFR absence type, after the screening amplification, recombinaant CHO cell strain efficient, stably express people BMP2 does not appear in the newspapers as yet.
Summary of the invention
Purpose of the present invention mainly is to provide a kind of recombinaant CHO cell strain CHO (rhBMP-2) that can efficiently express human bone morphorgenetic protein 2.
Another object of the present invention is to set up a technology platform that might be applied to express the other biological activated protein by setting up the method for recombinant C HO (rhBMP-2) cell strain.
A further object of the present invention has been to disclose recombinaant CHO cell strain CHO (rhBMP-2) and has had the application of inducing aspect the active and promotion bone defect repair of bone forming in the preparation treatment.Particularly the recombinaant CHO cell strain CHO (rhBMP-2) of Biao Daing induces the dystopy bone forming and promotes in external evoked bone forming, body has proved to have the obvious treatment effect aspect the bone defect repair.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of recombinaant CHO cell strain of efficiently expressing recombinant human Delicious peptide 2, called after CHO (rhBMP-2), this cell strain be by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, September 12 2006 preservation time.The strain of classification name Chinese hamster ovary cell, preserving number is CGMCC NO.1813
The recombinaant CHO cell strain of efficiently expressing recombinant human Delicious peptide 2 of the present invention is characterized in that, mainly is to utilize Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr
-) as host cell, the cell strain of after screening, amplification, being stablized, efficiently expressing.It is that C2C12 expresses alkaline phosphatase that the secreting, expressing product of cell strain can efficiently be induced sarcoplast, obtains the strain of CHO (rhBMP-2) monoclonal cell, and its expression amount reaches 7.8 3ug/24h10
6Cell.
Expression vector of the present invention is not limited to specific expression vector, as long as it can be recombinated with described dna fragmentation, forms suitable expression plasmid and gets final product.Prokaryotic cell prokaryocyte heterologous gene expression system for example; The eukaryotic cell heterologous gene expression system; Yeast cell to express system or the like, preferred expression vector is the eukaryotic cell heterologous gene expression system; It can be recombinated with described dna fragmentation, is easy to synthesize, and can effectively be secreted in the nutrient solution.And can form the suitable expression plasmid that activated protein is arranged of expressing.
The construction process of the recombinaant CHO cell strain CHO (rhBMP-2) of recombinant human bone morphogenetic protein 2 of the present invention may further comprise the steps:
(1) the synthetic and polymerase chain amplification technique of artificial oligonucleotide, the cDNA of human cloning BMP2 full length sequence.
(2) cDNA of people BMP2 full length sequence is cloned among the carrier for expression of eukaryon pCDNA3.1 (+), makes up mammalian cell expression vector pCDNA3.1 (+)-BMP2.
(3) use positively charged ion plasmalogen lipofectine2000 with pCDNA3.1 (+)-BMP2 and pSV2-dhfr cotransfection CHO-dhfr
-
(4) with the selection substratum screening integration BMP2 of G418 and xanthoglobulin, thymus pyrimidine and glycine and the stable transfected cells strain of dhfr gene.
(5) progressively increase progressively the mode of methotrexate concentration, the copy number of amplifying target genes.
(6) detect rhBMP2 in the recombinaant CHO cell substratum with Westen Blot.
(7) from mix the clone, be separated to mono-clonal by limiting dilution assay, utilize the expression amount of rhBMP2 in the ELISA method detection mono-clonal substratum, screen the engineering cell strain of highly effective expressing rhBMP 2.
(8) in the engineering cell strain culturing process secreting, expressing rhBMP2 in substratum.
Concrete preparation method of the present invention is described below:
1. test materials and method
1.1. cell cultures
Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell CHO-dhfr
-(CRL-9096; ATCC),, and add 10 therein with IMDM culture medium culturing (GIBCO company) available from Chinese Academy of Sciences's Shanghai biological chemistry and RESEARCH ON CELL-BIOLOGY institute
-4The xanthoglobulin of mol/L and 1.6 * 10
-5The thymidine of mol/L (all available from Sigma company) and 10% foetal calf serum of dialysing (available from Tianjin Hao ocean biological products company limited).The cell that this experiment relates to all is to contain 5%CO
2, cultivate in 37 ℃ the cell culture incubator.
1.2 the structure of eukaryon expression plasmid:
Original plasmid pCDNA6A-hBMP-2 that contains hBMP-2 cDNA full length sequence (GenBank accessionNo.NM001200) that has made up is a template with this laboratory, obtain the cDNA of full length sequence hBMP-2 by PCR reaction amplification, and be connected among the expression vector plasmid pCDNA3.1 (+), made up eukaryon expression plasmid pCDNA3.1 (+)-hBMP-2.For convenient clone, in the upstream and downstream primer, introduce BamHI and XhoI restriction enzyme site respectively.
Upstream primer: 5 '-GCTGGATCCACCATGGTGGCCGGGACCCGCTGTC-3 '
Downstream primer: 5 '-GCGTCTCGAGCTAGCGACACCCACAACCCTCCAC-3 '
The product transformed competence colibacillus cell E.coli DH5 α of ligation adopts the method for bacterium colony PCR to screen positive bacterium colony, and further extracts plasmid DNA and carry out enzyme and cut evaluation, and qualification result coincide with expection, and sequencing result shows that reading frame is entirely true.
1.3. transfection and screening positive clone
Plasmid pCDNA3.1 (+)-hBMP-2 and the pSV2-dhfr that build are mixed according to mol ratio at 5: 1, Lipofectamine2000 (available from Invitrogen company) cotransfection CHO-dhfr-cell is used unloaded plasmid pCDNA3.1 (+) and pSV2-dhfr cotransfection CHO-dhfr-cell in contrast simultaneously.Change fresh IMDM behind the cell transfecting 24h and select substratum (containing 10% FBS, the G418 of 700 μ g/ml) to cultivate, changed a not good liquor, and formed in per three days until cell clone.
1.4. the pressurization of methotrexate is cultivated:
All clones that form in the step 1.3 are digested with cell dissociation buffer Trypsin-EDTA (available from GIBCO company), be inoculated into mixed culture in the Tissue Culture Dish, after treating that cell growth reaches 90% density, be inoculated at 1: 6 in the new Tissue Culture Dish, and in selecting substratum, add methotrexate (MTX) (available from Sigma company), make its final concentration reach 0.1 μ mol/L, visible resistance clone occurs after about 2 week, all clones are digested, mixed culture, after cell growth condition is normal, again with the MTX amplification transformant of same program with 1 μ mol/L, finally obtain can be in the MTX of 1 μ mol/L the mixing clone strain of the recombinaant CHO cell of normal growth, called after CHO (rhBMP-2).
1.5.Western blot identifies rhBMP-2
CHO (rhBMP-2) is inoculated in the Tissue Culture Dish, treat that cell reaches 90% density after, use serum free medium instead and cultivate (not adding MTX), collecting cell nutrient solution after 2 days.Adopt the albumen in the trichloroacetic acid precipitation method extraction cell culture fluid.The albumen that extracts does the Tris-Tricine polyacrylamide gel electrophoresis and Western blot carries out Analysis and Identification.Sample is used the sample loading buffer mixing that contains DTT and do not contain DTT respectively, on 8% polyacrylamide gel, carry out electrophoresis together with Rainbow Marker, electrotransfer spends the night with the TBST damping fluid sealing that contains 5% skimmed milk to nitrocellulose filter then.Use an anti-anti-MP-2 (available from R﹠amp respectively; D company) monoclonal antibody is hatched 2h with the two anti-anti-mouse IgG (available from Promega company) that have horseradish peroxidase, then with ECL reagent react and exposure.
1.6.rhBMP-2 expression amount detect
With reference to " tissue culture and molecular cell learn a skill " described method CHO (rhBMP-2) cytomixis clone is carried out unicellular separation and Culture, obtained 14 strain monoclonal cell strains.This 14 strain monoclonal cell strain is inoculated into 96 orifice plates, cell growth is changed fresh culture after reaching 90% density, collect the nutrient solution supernatant behind the 24h, detect the concentration of rhBMP-2 in the nutrient solution supernatant with hBMP-2ELISA Kit (available from Wuhan doctor's moral company), use CCK-8 test kit (available from Japanese colleague's chemistry institute) to measure each porocyte number simultaneously, calculate the rhBMP-2 expression amount of each monoclonal cell strain according to the result.
1.7.rhBMP-2 biologic activity detect
It is C2C12 under the stimulation of BMP-2 is cultivated that bibliographical information mouse sarcoplast is arranged, and is transformed into scleroblast by the sarcoplast differentiation, and alkaline phosphatase (alkaline phosphatase ALP) is osteoblastic marker enzyme.We stimulate cultivation C2C12 with the rhBMP-2 conditioned medium of different concns, analyze the biologic activity of rhBMP-2 by the activity change of measuring ALP.
1.7.1 the preparation of conditioned medium
CHO (rhBMP-2) is inoculated in the IMDM substratum that contains 10%FBS and 1 μ mol/L MTX, after treating that cell growth reaches 90% density, use the DMEM that does not contain serum instead and cultivate (not adding MTX), collecting cell nutrient solution behind the 24h, 4000g, 4 ℃ of centrifugal 5min get supernatant and are sub-packed in the 1.5ml centrifuge tube,-70 ℃ of preservations are mixed in proportion use with normal substratum during use.
1.7.2 the mensuration of alkaline phosphatase activities:
CHO (rhBMP-2) the nutrient solution supernatant of collecting is added among the DMEM with 5%, 10% and 20% ratio respectively, and add FBS simultaneously, make the final concentration of FBS reach 5%, cultivate the C2C12 cell with this mixed culture medium, and cultivate the C2C12 cell in contrast with the DMEM that only contains 5%FBS, changed a not good liquor in per two days.Stimulate and cultivate after 5 days, earlier detect each hole in the O.D at 450nm place value with CCK-8 reagent, again with PBS damping fluid washing 3 times, every hole adds 50 μ l alkaline phosphatase enzyme reaction substrate solution, and (4 Sigma#104phosphatasesubstrate are dissolved in 15mL 50mmol/L glycine, 1mmol/L MgCl2, pH10.5), incubated at room 20min.Measure O.D value with Microplate Reader at the 405nm wavelength, the result divided by the O.D value of using CCK-8 reagent mensuration with standardized A LP result.Every group of sample got the mean value mapping in 6 multiple holes.
2. result
2.1 rhBMP-2 Construction of eukaryotic and evaluation:
With plasmid pCDNA6A-hBMP-2 is template, carries out PCR reaction, and amplification hBMP-2 cDNA inserts the PCR product among the plasmid pCDNA3.1 (+) again, obtain hBMP-2 carrier for expression of eukaryon pCDNA3.1 (+)-hBMP-2 (Fig. 1, Fig1).The plasmid that extracts is carried out digestion with restriction enzyme, and electrophoresis can obtain the segment of about 1.2kb/5.4kb size, and conforming to expected results, (Fig. 2, Fig2), sequencing result confirms that the segmental base sequence of insertion and reading frame are entirely true.
2.2 Western blot identifies the expression of rhBMP-2 in Chinese hamster ovary celI:
In Tissue Culture Dish, cell growth reaches and is replaced with the DMEM that does not contain serum after 90% the density and continues to cultivate, and collects nutrient solution behind the 48h with CHO (rhBMP-2) cell inoculation, and the trichloroacetic acid precipitation method is extracted nutrient solution albumen.With the protein sample that the extracts sample loading buffer mixing that contains DTT and do not contain DTT, and Tris-Tricine electrophoresis and Western blot evaluation (Fig. 3, Fig3).The result is presented at and occurs big or small 18kDa and big or small two the specific proteins bands that are about 30kDa of being about in reduction and the non-reducing sample lane respectively, shows CHO (rhBMP-2) secreting, expressing rhBMP-2 albumen.Amino acid according to hBMP-2 is formed calculating as can be known, be not about 13kDa through the rhBMP-2 albumen size of posttranslational modification, infer thus the rhBMP-2 that expresses be through posttranslational modifications such as glycosylation and be secreted in the substratum with the form of homodimer.
2.3 the expression amount of rhBMP-2 detects
Obtained the monoclonal cell strain of 14 strain rCHO (hBMP-2) by the method for unicellular separation and Culture, the ELISA method detects its expression amount.The result shows that expression amount can reach 3.6-7.83 μ g/24h/106cells.
2.4 the biologic activity of rhBMP-2 detects
Adopt contain respectively 5%, 10% and the conditioned medium of 20%CHO (rhBMP-2) cell 24h nutrient solution stimulate and cultivate C2C12, and cultivate the C2C12 cell in contrast with the DMEM that only contains 5%FBS, detection of alkaline phosphatase activity after 5 days (Fig. 4, Fig4).The result shows, the C2C12 cell neutral and alkali phosphatase activity that the conditioned medium made from CHO (rhBMP-2) cell culture fluid is cultivated has been compared remarkable height with control group, and show tangible dose dependent, this shows that the rhBMP-2 of expression has very strong biologic activity.Beneficial effect of the present invention is:
On the research basis of analysis-by-synthesis forefathers to Delicious peptide 2, be expression system with the Chinese hamster ovary cell of Tetrahydrofolate dehydrogenase defective type, by gene recombination technology, set up the engineering cell strain of stability and high efficiency express recombinant people BMP2.And have significant induced osteogenesis activity, recombinant human B MP2 by the recombinant human B MP2 of activity test proof recombinaant CHO cell strain secreting, expressing.The rhBMP2 that the recombinaant CHO cell strain of setting up by present method is expressed has physico-chemical property more similar to natural human BMP2 and biological activity, can be used for that the different clinically reasons of treatment cause that bone is damaged, diseases such as nonunion and fracture delayed union.
Description of drawings
The structure schema of Fig. 1 hBMP-2 expression vector pcDNA3.1 (+)-hBMP-2.
Fig. 2 pCDNA3.1 (+)-BMP2 makes up checking.Wherein A:hBMP2 D:pCDNA3.1 (+)-BMP2 enzyme is cut evaluation.
The Western Blot of Fig. 3 cho cell expressing recombinant rhBMP2 identifies.Wherein M is a molecular weight; 1 is contrast; 2 non-reduced conditions; 3 is reductive condition.
The biologic activity analysis of Fig. 4 recombinant human B MP2.ALP/CCK-8 wherein: relative reactivity rhBMP2 CM:CHO (BMP2) conditioned medium of alkaline phosphatase.
Embodiment
The present invention is described further below in conjunction with Figure of description and following specific embodiment, and embodiment only is indicative, means that never it limits the scope of the invention by any way.
Embodiment 1:pCDNA3.1 (+)-BMP2 makes up
According to the gene order of human bone morphorgenetic protein 2 (BMP2, SEQ ID NO.1), design and synthesize 2 pairs of special primers (B2F, B2R).With B2F, B2R is a primer, utilizes polymerase chain reaction (PCR) amplification to obtain the fragment (Fig. 2 A) of about 1200bp size.This fragment cloning is obtained pCDNA3.1 (+)-BMP2 in carrier for expression of eukaryon, through enzyme cut (Fig. 2 B), order-checking confirms consistent with the intended purposes sequence.
B2F：5’-gctggatccaccatggtggccgggacccgctgtc-3’
B2R：5’-gcgtctcgagctagcgacacccacaaccctccac-3’
Embodiment 2: the foundation of the engineering cell strain of stability and high efficiency express recombinant people BMP2
Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr-) is with the perfect medium that adds glycine, xanthoglobulin, thymus pyrimidine 10%FBS IMEDM (Hyclone), merges about 37 ℃, 5%CO2,10cm culture plate culturing cell to 80%.With positively charged ion plasmalogen Lipofectine 2000 (Invitrogen) with pCDNA3.1 (+)-BMP2 and pSV2-DHFR with 10: 1 ratios, according to the transfection of operational manual method, then with the selection substratum screening neo and the dhfr positive colony that contain 700ug/ml G418 and 10%FCS IMDM.The positive colony that obtains is with 1 * 10
6/ 10ml cell inoculation is in the 10cm Tissue Culture Dish, progressively with the IMDM culture medium culturing cell that contains 100nM and 1uM methotrexate (MTX) and 10%FBS, with amplifying target genes, finally obtain can be in containing 1uM MTX substratum the reconstitution cell of normal growth.The cell culture fluid albumen that extracts reconstitution cell carries out Western Blot, detects reconstitution cell rhBMP2 protein expression situation (Fig. 3).To be inoculated in the 96 porocyte culture plates with 0.3-0.5 cells/well through the mixing clone after the 1uM MTX amplification, cultivate with 10%FBS DMEM.Treat cell when converging, collect substratum, the ELISA method detects the engineering cell strain rCHO (BMP2) that the screening stability and high efficiency is expressed rhBMP2.
Embodiment 3: the external evoked bone forming activation analysis of recombinant human B MP2
Sarcoplast is when to be C2C12 with the DMEM that contains 10%FBS be cultured to 70-80% and converge, and PBS washing, trysinization, counting are with 2 * 10
4Individual cells/well is inoculated in the 48 porocyte culture plates, places 37 ℃, 5%CO2 incubator to hatch.The substratum that exhausts after 24 hours with PBS flushing twice, adds respectively and contains 5%, 10%, the conditioned medium of 20%rCHO (BMP2) 24h cell culture fluid stimulates and cultivate C2C12.Count every porocyte number with the CCK-8 test kit after 5 days, colorimetry is surveyed alkaline phosphatase activities (Fig. 4) then, and expression amount reaches as high as 7.83 μ g/24h/10
6Cell.
Sequence table:
＜110〉Nankai University
＜120〉the Chinese hamster ovary celI strain and the establishment method thereof of highly effective expressing rhBMP 2
<160>1
<210>1
<211>1547bp
<212>DNA
＜213〉people (Homo sapiens)
<400>1
ggggacttct?tgaacttgca?gggagaataa?cttgcgcacc?ccactttgcg?ccggtgcctt 60
tgccccagcg?gagcctgctt?cgccatctcc?gagccccacc?gcccctccac?tcctcggcct?120
tgcccgacac?tgagacgctg?ttcccagcgt?gaaaagagag?actgcgcggc?cggcacccgg?180
gagaaggagg?aggcaaagaa?aaggaacgga?cattcggtcc?ttgcgccagg?tcctttgacc?240
agagtttttc?catgtggacg?ctctttcaat?ggacgtgtcc?ccgcgtgctt?cttagacgga?300
ctgcggtctc?ctaaaggtcg?accatggtgg?ccgggacccg?ctgtcttcta?gcgttgctgc?360
ttccccaggt?cctcctgggc?ggcgcggctg?gcctcgttcc?ggagctgggc?cgcaggaagt?420
tcgcggcggc?gtcgtcgggc?cgcccctcat?cccagccctc?tgacgaggtc?ctgagcgagt?480
tcgagttgcg?gctgctcagc?atgttcggcc?tgaaacagag?acccaccccc?agcagggacg 540
ccgtggtgcc?cccctacatg?ctagacctgt?atcgcaggca?ctcaggtcag?ccgggctcac 600
ccgccccaga?ccaccggttg?gagagggcag?ccagccgagc?caacactgtg?cgcagcttcc 660
accatgaaga?atctttggaa?gaactaccag?aaacgagtgg?gaaaacaacc?cggagattct 720
tctttaattt?aagttctatc?cccacggagg?agtttatcac?ctcagcagag?cttcaggttt 780
tccgagaaca?gatgcaagat?gctttaggaa?acaatagcag?tttccatcac?cgaattaata 840
tttatgaaat?cataaaacct?gcaacagcca?actcgaaatt?ccccgtgacc?agacttttgg 900
acaccaggtt?ggtgaatcag?aatgcaagca?ggtgggaaag?ttttgatgtc?acccccgctg 960
tgatgcggtg?gactgcacag?ggacacgcca?accatggatt?cgtggtggaa?gtggcccact?1020
tggaggagaa?acaaggtgtc?tccaagagac?atgttaggat?aagcaggtct?ttgcaccaag?1080
atgaacacag?ctggtcacag?ataaggccat?tgctagtaac?ttttggccat?gatggaaaag?1140
ggcatcctct?ccacaaaaga?gaaaaacgtc?aagccaaaca?caaacagcgg?aaacgcctta?1200
agtccagctg?taagagacac?cctttgtacg?tggacttcag?tgacgtgggg?tggaatgact?1260
ggattgtggc?tcccccgggg?tatcacgcct?tttactgcca?cggagaatgc?ccttttcctc?1320
tggctgatca?tctgaactcc?actaatcatg?ccattgttca?gacgttggtc?aactctgtta?1380
actctaagat?tcctaaggca?tgctgtgtcc?cgacagaact?cagtgctatc?tcgatgctgt?1440
accttgacga?gaatgaaaag?gttgtattaa?agaactatca?ggacatggtt?gtggagggtt?1500
gtgggtgtcg?ctagtacagc?aaaattaaat?acataaatat?atatata 1547
Claims (5)
1, its preserving number of recombinaant CHO cell strain CHO (rhBMP-2) of efficiently expressing recombinant human Delicious peptide 2 is CGMCC NO.1813.
2, the recombinaant CHO cell strain of efficiently expressing recombinant human Delicious peptide 2 is characterized in that, utilizes Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr
-) as host cell, the cell strain of after screening, amplification, being stablized, efficiently expressing; It is that C2C12 expresses alkaline phosphatase that the secreting, expressing product of this cell strain can efficiently be induced sarcoplast, obtains the strain of CHO (rhBMP-2) monoclonal cell, and its expression amount reaches 7.83ug/24h10
6Cell.
3, the described recombinaant CHO cell strain of claim 1～2 CHO (rhBMP-2) has the application of inducing aspect the active and promotion bone defect repair of bone forming in preparation treatment.
4, the establishment method of claim 1 or 2 described CHO (rhBMP-2) cell strains may further comprise the steps:
(1) the synthetic and polymerase chain amplification technique of artificial oligonucleotide, the cDNA of human cloning BMP2 full length sequence;
(2) cDNA of people BMP2 full length sequence is cloned among the carrier for expression of eukaryon pCDNA3.1 (+), makes up mammalian cell expression vector pCDNA3.1 (+)-BMP2;
(3) use positively charged ion plasmalogen lipofectine2000 with pCDNA3.1 (+)-BMP2 and pSV2-dhfr cotransfection CHO-dhfr
-
(4) with the selection substratum screening integration BMP2 of G418 and xanthoglobulin, thymus pyrimidine and glycine and the stable transfected cells strain of dhfr gene;
(5) progressively increase progressively the mode of methotrexate concentration, the copy number of amplifying target genes;
(6) detect rhBMP2 in the recombinaant CHO cell substratum with Westen Blot;
(7) from mix the clone, be separated to mono-clonal by limiting dilution assay, utilize the expression amount of rhBMP2 in the ELISA method detection mono-clonal substratum, screen the engineering cell strain of highly effective expressing rhBMP 2;
(8) in the engineering cell strain culturing process secreting, expressing rhBMP2 in substratum.
5, the recombinaant CHO cell strain CHO (rhBMP2) that expresses of the described methods of claims 4 induces the dystopy bone forming and promotes application aspect the bone defect repair in the external evoked bone forming of preparation treatment, body.
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Cited By (9)
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| CN101831432A (en) * | 2010-04-19 | 2010-09-15 | 浙江理工大学 | Biosynthesis method and application of bone morphogenetic protein-2 |
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| CN102965342A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | Chinese hamster ovary gene engineering cell strain with high-efficient secretory expression of leptin |
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| CN113350486A (en) * | 2021-07-06 | 2021-09-07 | 深圳市人民医院 | Pharmaceutical composition with bone defect repairing effect and application thereof |
| CN117384959A (en) * | 2023-12-05 | 2024-01-12 | 南京东万生物技术有限公司 | Construction of collagen production cell strain and production method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101903526B (en) * | 2007-12-21 | 2014-02-12 | 诺华股份有限公司 | Organic compounds |
| CN101831432A (en) * | 2010-04-19 | 2010-09-15 | 浙江理工大学 | Biosynthesis method and application of bone morphogenetic protein-2 |
| CN101831432B (en) * | 2010-04-19 | 2012-10-03 | 浙江理工大学 | Biosynthesis method and application of bone morphogenetic protein-2 |
| CN102358893A (en) * | 2011-10-10 | 2012-02-22 | 黑龙江大学 | Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode |
| CN102965342A (en) * | 2012-12-12 | 2013-03-13 | 黑龙江大学 | Chinese hamster ovary gene engineering cell strain with high-efficient secretory expression of leptin |
| CN102965342B (en) * | 2012-12-12 | 2014-06-25 | 黑龙江大学 | Chinese hamster ovary gene engineering cell strain with high-efficient secretory expression of leptin |
| JP2019122303A (en) * | 2018-01-17 | 2019-07-25 | 東ソー株式会社 | Alkaline phosphatase high expression animal cell |
| JP7214962B2 (en) | 2018-01-17 | 2023-01-31 | 東ソー株式会社 | Alkaline phosphatase high expression animal cells |
| CN112996914A (en) * | 2018-09-04 | 2021-06-18 | 阿莱尔特森特尔创新生物技术有限责任公司 | Vector VTvaf 17-based gene therapy |
| CN112501126A (en) * | 2020-12-03 | 2021-03-16 | 康立泰药业有限公司 | CHO-DHFR + cell strain and application thereof |
| CN112501126B (en) * | 2020-12-03 | 2023-04-07 | 康立泰生物医药(青岛)有限公司 | CHO-DHFR + cell strain and application thereof |
| CN113350486A (en) * | 2021-07-06 | 2021-09-07 | 深圳市人民医院 | Pharmaceutical composition with bone defect repairing effect and application thereof |
| CN117384959A (en) * | 2023-12-05 | 2024-01-12 | 南京东万生物技术有限公司 | Construction of collagen production cell strain and production method thereof |
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